Simultaneously, the lignin showed high purity (90.4%), high relative molecular weight , and excellent uniformity. Furthermore, the aromatic ring structure of lignin remained intact, consisting mainly of p-hydroxyphenyl, syringl, and guaiacyl subunits. The lignin generated a large number of volatile organic compounds during the depolymerization process, mainly composed of ketones, phenols, syringols, guaiacols, esters, and aromatic compounds. Finally, the antioxidant activity of the lignin sample was evaluated with the 1,1-diphenyl-2-picrylhydrazyl radical scavenging assay; the lignin from water chestnut shells showed excellent antioxidant activity. These findings confirm that lignin from water chestnut shells has a broad application prospect in valuable chemicals, biofuels and bio-functional materials.The molecular weight of lignin extracted from lignocellulosic biomass is an important factor in determining its valorization in industrial processes. Herein, this work aims to explore the extraction of high molecular weight and bioactive lignin from water chestnut shells under mild conditions. Five kinds of deep eutectic solvents were prepared and applied to isolate lignin from water chestnut shells. The extracted lignin was further characterized with element analysis, gel permeation chromatography, and Ultraviolet-visible and Fourier-transform infrared spectroscopy. The distribution of pyrolysis products was identified and quantified with thermogravimetric analysis—Fourier-transform infrared spectroscopy and pyrolysis-gas chromatograph-mass spectrometry. The results showed that choline chloride/ethylene glycol/ The water chestnut is widely cultivated in several provinces in China. Water chestnut shells (WCSs), as the main by-product in water chestnut production, are mostly incinerated or discarded, resulting in environmental pollution and resource waste. Polysaccharides and polyphenols contained in WCSs have been developed and applied in the pharmaceutical industry, but their high lignin content has been neglected. Furthermore, the natural structural properties and high-value applications of lignin obtained from WCSs have not been fully investigated. Lignin is an aromatic polymer composed of a variety of chemical bonds linked to phenylpropanol derivatives, containing a variety of reactive functional groups, which can be converted into fuels, chemicals, and bio-functional materials . The keyO-4 and C–C bonds in the original lignin are destroyed during the treatment, resulting in low molecular weight and condensation reactions; these are not conducive to the high-value utilization of lignin in later stage [DES is a liquid eutectic mixture formed by mixing hydrogen bond donors (HBDs) and hydrogen bond acceptors (HBAs) at a certain molar ratio . Accordier stage .O-4 in the lignin structure. So far, the ball milling and γ-valerolactone (GVL)/H2O solvent methods can preserve the natural lignin structure of lignocellulose biomass, but the extraction efficiency is low [O-4 bonds in the lignin structure are depolymerized during the process [O-4 ether linkage retention of 61% [The molecular weight of lignin directly affects its physicochemical properties. Lignin with low molecular weight and high hydroxyl content is suitable for the preparation of bioactive materials , while ly is low ,18. Alth process . Provostn of 61% . To maxiO-4 bonds [p-toluenesulfonic acid as a solvent showed good lignin removal ability under mild conditions, and the recovered lignin had high purity and moderate molecular weight [p-toluenesulfonic acid (p-TsOH) as hydrogen bond donors. The separated lignin was characterized using an elemental analyzer, gel permeation chromatography (GPC), Ultraviolet-visible (UV-Vis) spectroscopy, Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TG)-FTIR, and pyrolysis-gas chromatograph-mass (Py-GC/MS) spectrometry. Its antioxidant activity was evaluated using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay. This study is expected to provide useful references for the high-value utilization of water chestnut processing waste and the development of bioactive functional materials.Based on previous research, lignin separated using carboxylic acid or alcohol-based DESs can retain a large number of β--4 bonds . In addir weight ,23. To op-toluenesulfonic acid (ChCl/EG/p-TsOH). This result may be due to the presence of active protons in an acidic environment, which may break the ether bonds between the lignin and carbohydrate complexes, thus making the lignin more soluble. However, alcohol-based DESs lack active protons and acidic sites [p-TsOH has a relatively low viscosity among DESs. Many studies have demonstrated that low-viscosity DESs penetrate more readily into plant cells to remove lignin and hemicellulose [This study focused on obtaining lignin with high purity and high bioactivity for the future development of lignin biorefined chemicals. Herein, lignin was extracted from WCSs using five ChCl-based DESs under mild conditions. The results are illustrated in ic sites . Additioellulose . The purO-4 content. The average molecular weight of the lignin isolated using ChCl/OA was lower than 2900 g/mol, probably due to the disruption of ether bonds in the structure of the lignin during extraction with DES, thus decreasing its molecular weight. However, the average molecular weight of the lignin separated using the DESs containing ethylene glycol was higher than 11,000 g/mol, while that of the lignin extracted using ChCl/EG/p-TsOH was the highest at 37,077 g/mol. This result may be because the main connecting bonds (ether bonds) in the lignin were not destroyed, thus preserving its original structure. Previous studies reported that the addition of ethylene glycol to DES can effectively preserve the β-O-4 bond in lignin and inhibit lignin condensation [p-TsOH [Further research on the lignin separated using the five kinds of DESs showed that the weight of lignin, i.e., the average molecular weight, varied greatly from 2884 to 37,077 g/mol. Those were related to the degree of lignin condensation and the β-ensation ,27. More [p-TsOH .p-TsOH, choline chloride/lactic acid (ChCl/LA), and choline chloride/ethylene glycol (ChCl/EG). However, when the lignin concentration exceeded 0.6 mg/mL, the DPPH radical-scavenging activity of the lignin obtained using carboxylic acid-based DES increased slowly and remained mainly between 80% and 89%. When the lignin concentration was 1.0 mg/mL, the lignin obtained using the various DESs showed a scavenging activity of 73.41–89.73% against DPPH radicals, with ChCl/OA > ChCl/LA > ChCl/EG/p-TsOH > ChCl/EG > choline chloride/glycerol (ChCl/GL). The lignin separated using the five kinds of DESs showed different scavenging activity against DPPH radicals, mainly due to differences in the content of phenolic hydroxyl in the lignin structure. According to previous reports, lignin with a high content of phenolic hydroxyl possessed the best antioxidant ability [Lignin contains a large number of phenolic hydroxyl groups and has potential as a natural antioxidant. Many studies have found that the phenolic hydroxyl content in lignin has a direct relationship with the extraction method and the source of raw materials . Therefo ability ,33.p-TsOH DES had the highest molecular weight, the elemental composition, chemical structure, and pyrolysis products were further analyzed.In summary, under mild and short time conditions, lignin of high purity was obtained from WCSs with the separation method used in this study, without further purification in the subsequent process. The obtained lignin has good DPPH radical scavenging activity, indicating the potential to develop lignin-based functional materials. Since the lignin separated with ChCl/EG/p-TsOH DES were 52.81%, 3.76%, 41.43%, 0.03%, and 1.55%, respectively. The presence of small amounts of N and S is probably due to the presence of small amounts of protein. This result is similar to that of the reported lignin in sugarcane bagasse [9H7.69O5.29 [Lignin composition determines its chemical structure and heating value. The C, H, O, S, and N contents in the lignin separated with ChCl/EG/ bagasse . Accordi.69O5.29 . A highe.69O5.29 , the obt2O solution. Among those, two absorption peaks at 200–350 nm indicate that the substance is a phenolic compound. Usually, lignin has a typical absorption peak at both 240 nm and 280 nm due to the electron transfer from non-conjugated phenolic structures in aromatics [Ultraviolet-visible spectroscopy is a simple and effective tool for the qualitative determination of the phenolic hydroxyl groups in lignin. With structures such as benzene rings, hydroxyl, methoxy groups, and carboxyl, lignin shows obvious ultraviolet absorption. romatics .3, and -COOH; thus, its characteristic chemical structure can be intuitively reflected in FTIR spectra. The FTIR spectrum for the lignin isolated with ChCl/EG/p-TsOH is shown in −1 was assigned to the stretching vibration in the aromatic and aliphatic hydroxyl groups. The absorption peak at 2943 cm−1 was attributed to the symmetric C–H stretch in the alkyl group. The absorption peak at 1718 cm−1 was attributed to the C=O stretching vibration in nonconjugated ketones and carbonyl groups, indicating that some benzene rings were destroyed during the extraction of lignin with DES, forming a quinoid structure. The absorption peak at 1616 cm−1 was assigned to the C=C conjugation stretch in the aromatic rings of coniferyl and sinapyl alcohols. The absorption peak at 1513 cm−1 was assigned to the vibration in the aromatic skeleton of lignin. The absorption peak at 1452 cm−1 was assigned to the asymmetric C–H deformation of methyl or methylene in the ether structure of guaiacyl, syringyl, and p-hydroxyphenylpropane (GSH)-type lignin. These absorption peaks confirmed that the benzene ring-based skeleton structure was well preserved during the extraction with DES. The absorption peak at 1336 cm−1 was assigned to the C–O groups corresponding to the syringl and guaiacol units. The peak at 1268 cm−1 was assigned to a guaiacyl unit and the C=O stretching vibration. The peak at 1212 cm−1 was ascribed to the C–O group. The peak at 1142 cm−1 corresponded to the aromatic ring of guaiacol and the C=O stretch. The peak at 1042 cm−1 corresponded to the C–H deformation of guaiacol, the C–O deformation in main alcohols, and the nonconjugated C=O stretch. Furthermore, there were no characteristic absorption peaks of carbohydrates at 1373 cm−1 and 1056 cm−1 [Lignin has various types of functional groups, such as -OH, -OCHp-TsOH), a thermal analysis was performed. The thermogravimetric (TG) and differential thermogravimetric (DTG) curves of the lignin are presented in Lignin is an organic polymer mainly composed of a phenylpropane unit and connected with various chemical bonds. The types of chemical bonds and functional groups directly affect the pyrolysis properties of lignin. To explore the thermal decomposition characteristics of the lignin extracted from WCSs with DES (ChCl/EG/M). Many studies have demonstrated that the TM of lignin during the pyrolysis was proportional to the number of β-O-4 linkages present in the molecule [p-TsOH showed the highest mass loss between 370 °C and 336 °C in our previous report [p-TsOH-had a lower TM, indicating that there were more β-O-4 linkages in the lignin. Moreover, at higher temperatures (400 °C to 500 °C), the shoulder peak corresponded to the cleavage of the C–C bond in the lignin. It is inferred from the results of TG/DTG that the thermal behavior of lignin has an intrinsic relationship with its inherent structure.TG curves may reveal lignin weight loss throughout various decomposition processes, while DTG curves can record the related maximum decomposition temperature ; however, CH4 was not found at 70 °C. This result is also in accordance with the TG analysis shown in 2, and CH4 during pyrolysis, with great potential to replace coal and natural gas as syngas feedstocks and raw materials for bioenergy production.Based on the results of TG and DTG, the change in volatiles with increasing temperature was also investigated. The FTIR spectra are depicted in The pyrolysis compounds, generated during the thermal degradation of the lignin, were separated using gas chromatography (GC) and identified using mass spectrometry (MS). The method facilitated an analysis of the structure and further exploration on the pyrolysis properties of the lignin, demonstrating the potential to transform lignin into high-value-added chemicals. The total ion chromatogram of the lignin is presented in O-4 bonds in the lignin. Similar results were previously mentioned by Zong et al. [According to their structures, these pyrolysis compounds were classified into guaiacol (G) types, syringol (S) types, catechol (C) types, aromatics, ketones, and esters. The presence of GSH-type compounds in the pyrolysis products indicated that the lignin from WCSs belonged to the GSH type, further verifying the results of FTIR. Although it is impossible to obtain the exact yield of each chemical in the pyrolysis products, the peak area of the molecule in the chromatographic relates linearly to its relative content, which can be utilized to reveal a relative content change in each identified product. The peak area of lignin pyrolysis products follows the order of G-type > aromatic > S-type > esters > H-type > ketones > C-type compounds. The G-type compounds were the main pyrolysis products, derived from the G-type unit and probably resulting from the breakage of many β-g et al. . In addig et al. . At the v/v) at 90 °C for 6 h in a Soxhlet extractor.Water chestnut shells, from Shandong province, China, were collected and ground into 80-mesh particle samples after washing and air-drying. To remove ethanol and water-soluble extracts, the samples were extracted with benzene/ethanol  were purchased from Aladdin Co., Shanghai, China. The used 2,2-Diphenyl-1-picrylhydrazyl (>98.5%) was purchased from Machlin Co., Shanghai, China.Choline chloride, ethylene glycol, p-TsOH) were mixed at different molar ratios and magnetically stirred at 80 °C until a transparent and homogeneous liquid was formed. The composition and corresponding molar ratios of the prepared DESs are summarized in The hydrogen bond acceptor (ChCl) and the hydrogen bond donors ,m denoted the lignin mass extracted from the WCSs and w denoted the total mass of lignin in the WCSs.The lignin extraction procedure is shown in The contents of acid-soluble lignin (ASL) and acid-insoluble lignin (AIL) in biomass or lignin samples were determined according to the methods used in the previous literature ,37. BaseThe lignin extract of different masses was dissolved in a 1,4-dioxane aqueous solution to obtain several lignin solutions of 0.2–1.0 mg/mL. To a 10 mL brown centrifuge tube were added 0.1 mL of the lignin solution and 3.9 mL of a DPPH ethanol solution (25 mg/L), respectively. Each mixed solution was shaken and stood in a water bath at 25 °C for 30 min, with the absorbance measured at 517 nm with UV-Vis spectroscopy.The viscosity of the DESs was determined at 30 °C using a viscometer . The surface tension of the DESs was measured using a contact angle meter .The elemental analysis  of the lignin samples was performed using a Vario EL cube elemental analyzer . The relative molecular weight distribution of the lignin sample was measured with GPC according to the methodology proposed by Alvarez-Vasco et al. .−1 with a resolution of 4 cm−1 and 32 scans.The UV-Vis absorption spectra of the lignin were recorded on an ultraviolet-visible spectrophotometer . About 5 mg of the lignin extract was dissolved in a 10 mL aqueous solution of 90% (v%) 1,4-dioxane. The absorbance was determined by scanning the solution over the wavelength range of 200–400 nm. Functional groups in the lignin were analyzed using FTIR . FTIR spectra were obtained in the range of 4000–400 cm2 atmosphere. Simultaneously, FTIR spectra of the volatiles were recorded in the region of 4000–400 cm−1.Thermal stability and volatiles generated during pyrolysis were carried out using a TG 209 F1 thermogravimetric analyzer  coupled with Nicolet iS 50 FTIR  at a temperature from 30 °C up to 800 °C at a heating rate of 10 °C/min under NThe pyrolysis process was performed with a PY-2020Id pyrolyzer  at 610 °C for 30 s. The pyrolytic products present in the lignin samples were estimated using QP2010 Ultra GC/MS  equipped with a UA-30M-0.25F capillary column (30 m × 0.25 mm × 0.25 μm) with high-purity helium as a carrier gas. The GC/MS operation parameters were an injector temperature of 300 °C, a helium flow of 1 mL/min, an increasing oven temperature from 50 °C to 280 °C with a rate of 8 °C/min, an electron ionization mode at 70 eV, and the m/z ranging from 30 to 550. The main pyrolytic products were identified using the NIST 11 library with a probability match (>80%) for the qualitative comparison of the products.p-TsOH could obtain a higher lignin extraction yield (84.17%). Simultaneously, the structure and composition of the extracted lignin were analyzed in detail using FTIR, TG-FTIR, and Py-GC/MS. The results demonstrated that the lignin separated with ChCl/EG/p-TsOH DES had the characteristics of high purity, high molecular weight , and low PDI (1.36) and relatively completely retained the original lignin structure. Furthermore, the pyrolysis characteristics of the lignin showed that it had good thermal stability and released a lot of volatile substances, such as ketones, phenols, syringols, guaiacols, esters, and aromatic compounds, during pyrolysis at high temperatures. When the lignin concentration was 1.0 mg/mL, the lignin obtained with various DESs showed a scavenging activity of 73.41–89.73% against DPPH radicals. The above results indicate that the lignin from WCSs has the potential as a high-value-added chemical.In this study, five kinds of DESs were synthesized, and the corresponding physicochemical properties were analyzed. Among these, the most effective DES was obtained by performing the isolation of lignin from the WCSs. Under mild conditions, the DES composed of ChCl/EG/


The lithium niobate microring resonator is fabricated by photolithography-assisted chemo-mechanical etching (PLACE) technique, and the Q factor of lithium niobate microring is measured as high as 6.91 × 105. The linewidth of the multimode 980 nm laser diode, which is ~2 nm measured from its output end, is narrowed down to 35 pm with a single-mode characteristic after coupling with the high-Q LN microring resonator. The output power of the narrow-linewidth microlaser is about 4.27 mW, and the wavelength tuning range reaches 2.57 nm. This work explores a hybrid integrated narrow linewidth 980 nm laser that has potential applications in high-efficient pump laser, optical tweezers, quantum information, as well as chip-based precision spectroscopy and metrology.We demonstrate a narrow linewidth 980 nm laser by self-injection locking of an electrically pumped distributed-feedback (DFB) laser diode to a high quality (Q) factor (>10 Combnd Si3N4 ,23,24. Hnd Si3N4 ,26. HoweThe reliable semiconductor 980 nm pump lasers are key components in ytterbium/erbium ions doped lasers and optical fiber amplifiers, which are critical to high-throughput laser material processing and optical fiber communication systems ,30. Pump2 and a low writing speed of ~1 cm2/hour, whereas the PLACE technique has a large writing field (8 inch) and a high writing speed (1 cm2/min), so the PLACE technique can easily fabricate the 4-inch LNOI wafer in 2 h in our lab. The footprint of a photonic chip fabricated by PLACE technique is only limited by either the wafer size or the motion range of the positioning stage, due to the unique advantage of femtosecond laser direct writing. Thus, the PLACE technique allows for the expansion of the footprint of photonic chip almost up to 0.5 × 0.5 m. In addition, the photonic structures fabricated by the chemo-mechanical polishing (CMP) process can have a surface roughness sub-nanometer, reducing the propagation loss approaching the absorption limit of the LN crystal [Here, a high-performance narrow-linewidth 980 nm laser has been demonstrated by self-injection locking of a seed laser to a high-Q lithium niobate (LN) microring resonator. The 980 nm laser diode source is a commercial Chip-on-Submount (CoS) laser. The linewidth of 980 nm COS laser diode is reduced from about 2 nm to 35 pm. We also observed the transition from multimode to single-mode operation by monitoring the lasing modes at the output of the LN microring resonator using an optical spectral analyzer. The output power of the narrow-linewidth laser is about 4.27 mW, and the narrow-linewidth laser can be tuned in a wavelength range of 2.57 nm. In this self-injection locking scheme, the LN microring is fabricated by the photolithography-assisted chemo-mechanical etching (PLACE) technique developed in our lab, whereas other high-Q WGM microresonators used in self-injection locking schemes are often fabricated by electron beam lithography (EBL) ,20,21. I crystal . These u crystal .2)/Silicon (Si) where the thicknesses of X-cut TFLN, SiO2 and Si were 500 nm, 4.7 µm and 525 µm, respectively. Secondly, as shown in ous work .pp) applied to the continuously tunable laser (CTL) to scan finely around 980 nm wavelength. At the same time, the electrical signal from the AFG was also linked to the OSC to trigger and synchronize the transmission spectrum channel. 5 (975.36 nm) and 6.53 × 105 (975.51 nm), respectively. The free spectral range (FSR) of the LN microring is 0.15 nm.As shown in δω is the linewidth of self-injection-locked DFB laser, freeδω is the linewidth of the free-running DFB laser, dQ (102) and Q (105) are the quality factors of the laser diode cavity and of the LN microring cavity, and gα is the phase-amplitude coupling factor. According to the formula, the order of magnitude of the linewidth compression factor is 106, and thus the theoretically calculated linewidth is 600 kHz (1.92 × 103 pm). However, the experimentally measured linewidth is 35 pm, which is much wider than the theoretical prediction. Actually, the measurement resolution of the linewidth of our self-injection locked laser diode is limited by the highest resolution of OSA, which is only 0.01 nm. The actual linewidth compression factor should be much greater than that measured by OSA, and the actual linewidth needs to be characterized with the self-delayed heterodyne system, which is currently not available in our laboratory. Such a measurement will be carried out in the future.As shown in As shown in 5. An OSA is used to characterize the linewidth of self-injection locked laser diode, which is narrowed down from about 2 to 35 pm (limited by the resolution of OSA). The transition from multimode to single mode can be observed clearly after a self-injection locked with LN microring resonator. The output power from the integrated narrow-linewidth laser diode is 4.27 mW, and the wavelength is tuned with a range of 2.57 nm. This narrow-linewidth 980 nm laser has potential applications in high-efficient pump lasers, optical tweezers, quantum information, as well as chip-based precision spectroscopy and metrology. Further improvement by fabrication of the spot size converter (SSC) on the LN microring chip both for lensed fiber and DFB laser, the spot size of input waveguide on LN microring chip must match the DFB laser diode, and the spot size of output waveguide on LN microring chip must match the fiber and as closely as possible to ensure good coupling. We can integrate microelectrodes near the LN microring resonator will enable continuous precious and high-speed tuning by electro-optic and thermal-optic effects in LN crystal. We will mount the self-injection locked laser on a thermoelectric refrigerating unit (TEC) for more stable laser emission, and TO-packaged can also be applied to the self-injection locked laser chip. The SiO2 cladding will be coated on LN microring chip to prevent contamination for stable high Q. More importantly, we will measure the linewidth accurately using the delayed self-heterodyne interferometer setup or high-quality laser linewidth/phase noise analyzer in the future.In conclusion, we demonstrate a high-performance narrow linewidth 980 nm laser by self-injection locking of a laser diode to a LN microring resonator of high-Q. The LN microring resonator is fabricated by PLACE technique which has a high-scale writing field, high writing speed, and sub-nanometer surfaces. The Q factor of lithium niobate microring is measured as high as 6.91 × 10


Globularia alypum L. (GA) influences inflammation and oxidative stress modulation in rats and in vitro. The present study aims to investigate the effects of this plant in patients with ulcerative colitis (UC) and normal controls. In our experiments, we pretreated colon biopsies from 46 UC patients and normal controls with GA leaves aqueous extract (GAAE) used at two concentrations (50 and 100 µg/mL) for 3 h, followed by Lipopolysaccharides (from Escherichia coli) stimulation. We analyzed the effects on inflammation by studying the cyclo-oxygenase-2, the intercellular adhesion molecule-1, the nuclear factor kappa B, and p38 mitogen-activated protein kinase expression. Moreover, we assessed the levels of interleukin 6, the superoxide dismutase activity, and nitric oxide release in the supernatant of cultures. Our data showed that GAAE influences UC patients and normal controls for most studied markers and enzymes. These results acknowledge, with some scientific evidence, the traditional belief in the anti-inflammatory properties of GA and represent the first demonstration of its effect in a human in vitro model of inflammatory conditions.The existing literature indicates that  Globularia alypum L. (GA) is a Mediterranean-growing plant in the Globulariaceae family [Mycobacterium tuberculosis [50 value in rats was found to be above 14.5 g/kg [e family . Historie family ,3,4. Some family . The acte family . More ree family ,7,8, whirculosis ,10,11,12rculosis . Anotherrculosis . GA workrculosis , and an 4.5 g/kg , and a 14.5 g/kg . DespiteAmong human diseases causing inflammation, UC is an idiopathic chronic inflammatory disease of the gastrointestinal tract . ColitisThe current work investigates some of the known anti-inflammatory and anti-oxidant capabilities and effects of GAAE in an ex vivo in vitro model of human gut colonic explants from UC patients and controls.g), the resulting GAAE was lyophilized and kept at −80 °C [GA was gathered in March 2018 from the region of Boussalem (north-west Tunisia). This specimen has been cataloged as GA-ISBB-03/03/18 and is housed in the herbarium of the Higher Institute of Biotechnology of Beja at Jendouba University. For 72 h at 40 °C in an incubator, GA leaves  were dried and then ground in an electric mixer. The plant powder was then added to distilled water, and the mixture was incubated at room temperature for 24 h with magnetic stirring. After centrifugation .The cultured intestinal explants provide an ex vivo in vitro model that faithfully recapitulates the intestinal environment down to the individual cell populations and their interdependencies.The biopsies were put villous-side up on a stainless steel mesh and then placed above the central well of an organ culture dish . The biopsies’ slicing surfaces were brought into contact with the culture medium by adding it to each well. DMEM F12 16 mL), fetal calf serum (3 mL), 50,000 IU penicillin, and 5000 IU streptomycin made up the culture medium. At 37 degrees Celsius, the dishes were gassed with 95% oxygen and 5% carbon dioxide in an anaerobic jar [ mL, fetaEscherichia coli-lipopolysaccharides (EC-LPS) and left to sit overnight. We terminated all cultures after the first day. For further analysis, the tissue was flash-frozen in liquid nitrogen and kept at −80 °C.One well contained just culture medium as a negative control, four wells were pretreated with GAAE (50 and 100 µg/mL), and one well served as a positive control. After 3 h of incubation, each culture was given 1 µg/mL of All the cultured biopsies obtained from the 31 UC and 15 control colons were tested by immunohistochemistry. Cryostat 5 µm thick mucosal slices were created and utilized for immunological labeling. Sections were fixed in acetone for 15 min before being treated separately with the following antibodies: human anti-COX-2 , human anti-ICAM-1 , Human p38 MAPK . However, for the NF-κB staining, after being fixed in 4% formaldehyde for 15 min, sections were permeabilized for 5 min with Triton and washed three times in PBS. Then, they were incubated in block buffer  for one hour. The human anti-NF-κB antibody  was added and incubated overnight.After the first incubation, all sections were washed three times with PBS. Alexa Fluor 488 conjugated anti-mouse IgG was added to the sections for 60 min at room temperature, and the sections were then washed three times with PBS. The tissue sections were then put in a solution of DAPI (1:1000) for 5 min, after which they were mounted with a solution of 20% glycerol in PBS. A fluorescence examination with a Nikon eclipse was used to examine the data. Object 20X and software for processing images were used to take the pictures.Samples of the intestine were broken up in RIPA buffer in the presence of protease and phosphatase inhibitors. The Bradford  method measured the amount of proteins in the supernatant. The concentration of proteins was calculated using a standard range of BSA with known concentrations. From each biopsy, 40 μg of total proteins are adjusted with lysis buffer (RIPA), mixed with 10 μL of LAEMMLI buffer, and then denatured at 95 °C for 5 min. Ten percent of sodium dodecyl sulfate/polyacrylamide gel electrophoresis was used to put the lysates on the gel (SDS-PAGE).The migration was performed at 80 V and subsequently at 120 V. The proteins were then transferred from the gel to a nitrocellulose membrane using a transfer cassette  and incubated for one hour at room temperature with a blocking solution (5% dehydrated milk powder dissolved in TBST). Monoclonal antibodies against COX-2, NF-κB, ICAM-1, and p38 MAPK were applied to membranes overnight at 4 °C. Following three TBST buffer washes, the membranes were treated for one hour with horseradish peroxidase-linked goat anti-mouse or anti-rabbit secondary antibodies (1:1000). The rabbit antibody-actin and lamin A/C were used as internal controls . The Chemidoc was used to visualize the findings of Western blot detection reagents .As previously described , nitric The SOD activity was assessed using the Misra and Fridovich technique , which iThe ELISA method was used to measure the amounts of IL-6 in the supernatant, and a commercial kit was utilized for the measurement . The protein levels were determined by employing a microplate reader with the 450 nm wavelength setting . The amounts of protein were adjusted such that they were equivalent to the conventional levels of protein.t-test for paired and unpaired data when necessary. Values were considered statistically significant at p < 0.05.Data are presented as means SEM and were carried out using Student’s The expression of ICAM-1 in situ  was greaThe COX-2 in situ expression is shown in Most controls in situ B have deThe EC-LPS challenge caused an increase in the levels of IL-6 in the biopsies. Still, this rise was suppressed when the colons were pretreated with either 50 or 100 µg/mL of GAAE . The levAfter EC-LPS stimulation, there was a discernible rise in the measured enzyme activity. This activity decreased after GAAE pre-treatment, especially with 100 µg/mL.The EC-LPS stimulation strongly increased NO production in all biopsies . The preResults from the immunohistochemistry of biopsies from UC patients showed a more interesting effect of EC-LPS on COX-2 and ICAM-1 expression , respectWestern blotting results confirmed the in situ results F and proIn UC patients, NF-κB and p38 MAPK expressions were mostly higher in patients under EC-LPS stimulation B. On theThe determination of the expression level of both NF-κB and p38 MAPK was also performed by Western blotting, which proved the antagonist effect of GAAE, particularly on NF-κB expression F. The exIn the biopsies derived from UC patients , we obseThe SOD activity was also evaluated from the biopsies of six UC patients . An incrThe EC-LPS stimulation increased NO production in most cultures . In thosThe increased interest in medicinal plant extracts in treating intestinal inflammatory diseases has prompted several clinical studies assessing the potential pharmacological properties of plants, their side effects, and costs .We investigated GAAE treatment in colon explants from controls, and UC patients challenged with EC-LPS, showing a significant control of the inflammation.In our setting, normal control colon biopsy cultures showed an increase in levels of NO, SOD activity, COX-2, NF-κB, ICAM-1, p38 MAPK, and IL-6 expression upon stimulation with EC-LPS. In addition, colon biopsies from UC patients showed an increase in NF-κB and COX-2 expression under the EC-LPS effect. However, in UC, the ICAM-1 and p38 MAPK, already increased for the disease-induced inflammation, were less affected by the EC-LPS exposure than in controls. The GAAE pre-treatment remarkably decreased COX-2 and NF-κB. In addition, IL-6 levels increased with EC-LPS exposure in UC and were partially inhibited after GAAE pre-treatment.Previous studies in healthy rats  and humaOur data indicate that GAAE pre-treatment reduces the activity of some of those markers in colon biopsies and further demonstrates that NF-κB is primarily involved in EC-LPS inflammatory activation. The observed high level of ICAM-1 before the UC biopsies stimulation may explain why the EC-LPS exposure had no high influence on the ICAM-1 level. On the other hand, GAAE inhibited ICAM-1 much more in normal biopsies than in UC samples. Therefore, GAAE seems to act preventatively against ICAM-1 expression more than curatively.The NF-κB is essential for producing pro-inflammatory genes, such as IL-6 . IL-6 isIn the present study, UC colon biopsy demonstrated an increase in oxidative stress, as evidenced by the up-regulation of SOD and NO production upon EC-LPS stimulation. The present findings are consistent with prior in vivo investigations in rats, utilizing GAAE as a therapy for acetic acid-induced colitis . The actOur data confirm the GAAE’s ability to scavenge ROS  and estaThe study limitations were the small number of individuals included and the limited number of biomarkers investigated. The number of individuals was initially greater than presented here. However, we had to exclude many cases because of inadequate culture conditions or the small size of biopsies, which limited the number of tests, giving incomplete results. As per the limited number of biomarkers assessed, we chose the biomarkers directly involved in illness manifestation and activation by EC-LPS. Other biomarkers, particularly cytokines, might be implicated; however, the size of the biopsies and the amount of the culture media, together with the several repeats for each test, limited the study’s ability to cover a broader range of tests.In conclusion, data from our pilot trial formulate the hypothesis that GAAE should be further investigated as an adjuvant therapy for UC/inflammatory bowel diseases, but more interestingly, to prevent chronic intestinal inflammation.


The effect of the combination of atezolizumab and bevacizumab (Atez/Bev) for hepatocellular carcinoma (HCC) on pulmonary arterial hypertension (PAH) is unknown. Estimation of PAH by using computed tomography (CT) has recently been proposed. Thus, we aimed to estimate the effect of Atez/Bev on PAH using CT. Altogether, 113 patients who received Atez/Bev for HCC were enrolled. Probable PAH was defined as the diameter of the main pulmonary artery (mPA-D) ≥ 33 mm, whereas suspicious PAH was defined as mPA-D ≥ 29 mm or mPA-D/the diameter of the ascending aorta (aAo-D) ≥ 1.0. Before treatment, probable/suspicious PAH were diagnosed in 7 (6.7%)/22 (21.0%) patients, respectively. mPA-D and mPA-D/aAo-D significantly increased after induction of Atez/Bev. The increment of mPA-D was correlated with the occurrence of post-treatment respiratory/heart failure. In analysis of 55 patients who underwent CT at 3 months after the last dose of Atez/Bev, mPA-D and mPA-D/aAo-D significantly decreased. However, in the group with continuous treatment of other molecular-targeted drugs after Atez/Bev, mPA-D and mPA-D/aAo-D showed no significant change. In conclusion, PAH may not be a rare complication in patients with HCC and should be managed carefully because of the possible negative effect of Atez/Bev on PAH. Portopulmonary hypertension (PoPH) is a subgroup in the clinical classifications of PAH4, and defined as PAH that is a consequence of portal hypertension often caused by liver diseases6.Pulmonary arterial hypertension (PAH) is a severe and progressive disease that is characterized by obliteration of pulmonary arteries, which leads to increased pulmonary vascular resistance and subsequent right heart failure7. Recently, the combination of atezolizumab, a fully humanized anti-programmed death-ligand 1 antibody, and bevacizumab, a vascular endothelial growth factor (VEGF) targeting antibody, (Atez/Bev) has been approved as the standard first-line systemic therapy for HCC, based on the IMbrave 150 trial9.Hepatocellular carcinoma (HCC), which is the most common type of primary liver cancer, is among the leading causes of cancer-related deaths worldwide10. VEGF and its tyrosine kinase receptor, VEGF receptor-2, play a central role in angiogenesis, protection of endothelial cell, and destabilization of endothelial barrier function of many respiratory disorders including PAH11. Contradictory reports suggest both protective and deleterious mechanisms on PAH by inhibition of VEGF signaling. VEGF blockade leads to endothelial cell apoptosis in most organ systems12, and the inhibition of VEGF signaling causes PAH by aggravating vascular remodeling14. Contrarily, VEGF reportedly protects the endothelial cell from injury and apoptosis, decreasing the risk of PAH15. Additionally, the use of Atez may promote cytotoxic lymphocytes and affect endothelial functions16. The prevalence of PoPH is reported to be 2–6% in patients with portal hypertension and 1–2% in patients with cirrhosis20. However, the prevalence of PAH in patients with HCC receiving chemotherapy is unknown, and the effect of Atez/Bev on PAH is largely unknown. Since HCC frequently occurs in patients with underlying chronic liver diseases, it is likely that some amount of PAH is present in patients with HCC, and its effect cannot be ignored clinically.Bev selectively binds to VEGF and inhibits angiogenesis and neovascularization, leading to the control of cancer cell proliferation and prevention of metastases21. In addition, the variation in the main pulmonary artery size over time is associated with progression in pulmonary arterial pressures and prognosis22.Right heart catheterization (RHC) is the gold standard for PAH assessment. However, it is not frequently performed due to its invasiveness. Recently, measurement of the main pulmonary artery and ascending aorta diameters using computed tomography (CT) has been reported to be useful in PoPH screeningAgainst these backgrounds, in the present study, we aimed to estimate the prevalence of PAH in HCC patients receiving systemic chemotherapy and to investigate the effect of Atez/Bev on PAH based on the change of pulmonary artery diameter.We retrospectively enrolled consecutive patients with HCC who were treated with Atez/Bev between October 2020 and December 2021. Clinical and radiological data were obtained from our institutional database. The treatment plan for HCC was finalized after the benefits and side effects of the various treatment modalities and recommendations from experts were explained. This study set out the following exclusion criteria: (1) patients with missing chest CT data after Atez/Bev administration; (2) patients with pulmonary, cardiogenic, and connective tissue diseases that may affect pulmonary hypertension.This study conformed to the principles stipulated in the Declaration of Helsinki and was approved by the Ethics Committee of Chiba University Graduate School of Medicine. The Ethics Committee of Chiba University Graduate School of Medicine approved that informed consent was waived because of the retrospective design.23.HCC was diagnosed using the American Association for the Study of Liver Disease criteria, and advanced HCC was defined as the presence of macrovascular invasion or extrahepatic metastasis. Portal vein tumor thrombosis (PVTT) was defined as a blood flow abnormality in the portal vein trunk with vascularization assessed by contrast imaging. The clinically recognizable PVTT is VP2-4, with a tumor thrombus above the second-order branches of the portal vein24, and Japanese guidelines propose that CT finding of mPA-D ≥ 33 mm might be helpful in the diagnosis of PAH2. According to the guidelines and a previous study24, in this study suspicious PAH was defined as the presence of mPA-D ≥ 29 mm or mPA-D/aAo-D ≥ 1.0, whereas probable PAH was defined as the presence of mPA-D ≥ 33 mm.Chest CT was taken at 5-mm slice thickness. The widest diameter of the main pulmonary artery (mPA-D) and ascending aorta (aAo-D) on the same slice level was measured. The mPA-D was selected within 3 cm of the bifurcation of the pulmonary artery. European guidelines suggest that CT findings of mPA-D ≥ 29 mm or mPA-D/aAo-D ≥ 1.0 might suspect pulmonary hypertensionChest CT before Atez/Bev administration and after 1 or 2 courses of Atez/Bev was performed. Moreover, chest CT data obtained before and at 3 months after the last dose of Atez/Bev was evaluated. Two experienced hepatologists, blinded to clinical data, measured the mPA-D and aAo-D.t-test, Mann–Whitney U-test, or paired t-test, as appropriate. Categorical variables were analyzed using Fisher’s exact, chi-squared test, or McNemar’s test, as appropriate. The multivariate analysis was assessed by logistic regression. The cumulative survival rate was calculated using the Kaplan–Meier method. The correlation coefficient (r) was assessed using Spearman’s rank correlation coefficient or Pearson correlation coefficient, as appropriate. Statistical data were analyzed using SAS version 9.2 , and P < 0.05 was considered statistically significant.Data are expressed as the mean ± standard deviation for normally distributed continuous variables, median and interquartile range for not normally distributed continuous variables, and frequencies and percentages for categorical variables. Continuous variables were analyzed using Student’s This study conformed to the principles of the Declaration of Helsinki and was approved by the Ethics Committee of Chiba University Graduate School of Medicine. The Ethics Committee of Chiba University Graduate School of Medicine approved that informed consent was waived because of the retrospective design.Among the 117 consecutive HCC patients who received Atez/Bev therapy during the study period, 105 patients were enrolled in the analysis after exclusion of 12 patients who met the exclusion criteria . Table Before the administration of Atez/Bev, the mean mPA-D, aAo-D, and mPA-D/aAo-D were 26.3 mm, 34.9 mm, and 0.76, respectively. The mPA-D ≥ 29 mm, mPA-D ≥ 33 mm, and mPA-D/aAo-D ≥ 1.0 were found in 21 (20.0%), 7 (6.7%), and 4 (3.8%) patients, respectively. Twenty- wo patients (21.0%) had suspicious PAH, and seven patients (6.7%) were diagnosed as having probable PAH.vs. suspicious PAH: 24.9 ± 2.9 vs. 31.7 ± 1.9 mm, P < 0.001; 0.72 ± 0.10 vs. 0.89 ± 0.11, P < 0.001, respectively). However, there was no significant difference in the aAo-D between the non-suspicious and suspicious PAH .The mPA-D and mPA-D/aAo-D were significantly larger in patients with suspicious PAH than those with non-suspicious . This was mirrored by an increased number of suspicious and probable PAH cases after Atez/Bev (pre-Atez/Bev vs. post-Atez/Bev: 22 (21.0%) vs. 31 (29.5%), P = 0.013; 7 (6.7%) vs. 13 (12.4%), P = 0.034, respectively) Contrarily, there was no significant difference in the aAo-D after Atez/Bev therapy between the two groups . High mPA-D level was the only predictive factor for the group that changed from non-suspicious PAH to suspicious PAH (P < 0.001). The increment of mPA-D was significantly correlated with advanced HCC  and older age .The decrease of mPA-D at 3 months after the last dose of Atez/Bev was significantly correlated with the presence of respiratory/heart failure  and the absence of continuous treatment of other molecular-targeted drugs after Atez/Bev . Contrarily, mPA-D and mPA-D/aAo-D significantly decreased in the group without continuous treatment of other molecular-targeted drugs .After the last dose of treatment, mPA-D and mPA-D/aAo-D significantly decreased  may be associated with the development or exacerbation of PAH, and dasatinib, a second-generation tyrosine kinase inhibitor, is being suggested as the causative agent of PAH28. The discontinuation of dasatinib and specific PAH therapy may lead to the resolution of PAH34. These are consistent with our results that discontinuation of Atez/Bev was associated with the decrease of mPA-D and mPA-D/aAo-D. Contrarily, continuous treatment using molecularly targeted drugs after the last dose of Atez/Bev therapy interfered with the improvement. These results suggest that VEGF inhibition during Atez/Bev therapy might have a negative effect PAH, and early diagnosis of PAH is important to determine whether Atez/Bev needs to be discontinued or a specific treatment for PAH needs to be added.Notably, the number of probable PAH cases based on the CT findings increased up to 12.4% after the induction of Atez/Bev. Treatment with Bev has been reported to increase the risk of many cardiovascular events38. Measurement of the tricuspid regurgitation pressure gradient values by echocardiography is used for the screening of PAH, especially when RHC could not be available35. Additionally, CT parameters, mPA-D and mPA-D/aAo-D, has been reported as reliable imaging markers for the screening of PAH, with relatively high sensitivity and specificity39. Although RHC is an essential test for the definitive diagnosis of PAH and for determining the efficacy of treatment36, the imaging-based assessment has significant advantages over RHC, as it is less invasive. In comparison with echocardiography, CT is frequently performed during systemic therapy for HCC; it is one of the essential tools for determining the efficacy of systemic therapy. Therefore, proactive use of CT as the first step modality to select candidate for the further examinations, such as echocardiography and RHC, is advocated because early screening for PAH leads to an early definitive diagnosis in patients with HCC.Regarding screening for PAH, recent advances in digital technology have improved the reliability of echocardiography-based screening for PAH, and such use of digital imaging has become increasingly popular41. Several studies have recently reported that female sex is related to the risk of pulmonary hypertension, which is consistent with the present results21. Genetic variation in aromatase and changes in estrogen metabolites have been reported to play an important role in the pathogenesis of PAH.It is well known that the idiopathic and familial forms of PAH occur more commonly in women than in men42. Therefore, caution should be exercised during the administration of Atez/Bev to elderly patients to avoid the development of side effects. Meanwhile, the latter finding may be explained by tumor-associated cytokines, which lead to further activation of macrophages and intimal proliferation, resulting in exacerbation of PAH43. This finding is also supported by data showing a relationship between advanced cancer and portal hypertension44. Although tumoral portal hypertension is reported to be a relatively rare condition in patients with HCC45, this condition would be a worthwhile area for future research, especially as the prognosis of advanced HCC has improved in recent years9.Interestingly, advanced age and advanced HCC were shown to be factors related to the increment of mPA-D and mPA-D/aAo-D after Atez/Bev treatment. Elderly patients reportedly have a higher likelihood of not being able to complete the trials due to toxicity and a high rate of adverse events when taking TKIs plus PD-1 inhibitorsWe expected that hypertension, a side effect of VEGF inhibitors, would cause an increment of aAo-D as well as contribute to the increment of mPA-D. However, hypertension did not contribute to either increment of mPA-D or aAo-D. This may be because the patients in the present study were given calcium channel blockers and/or angiotensin II receptor blockers early in the course of their treatment when the side effects of hypertension appeared.There are some limitations to our paper. First, the data were retrospectively analyzed at a single center. Therefore, the study is limited in its generalizability due to the possibility of population selection bias. Second, although a definitive diagnosis of PAH is based on echocardiography and RHC, this study has no data on RHC and echocardiography. Therefore, the impact of Atez/Bev on PAH should be examined in a prospective setting, taking into account the data on RHC and echocardiography.In conclusion, PAH may not be a rare complication in patients treated for HCC, and Atez/Bev treatment may have a possible negative impact on PAH. Moreover, in patients with advanced HCC and elderly patients, the increment of pulmonary artery diameter should be monitored carefully.


Unlike previous work, proteins were captured by an irreversible chemical reaction rather than by non-covalent affinity binding or physical entrapment. Because the protein-reactive group was attached to fluorescein, which in turn was coupled to the hydrogel by a photocleavable bond, on release the protein was labelled with fluorescein. Our hydrogel offered a pre-concentration factor of up to 236 for a model protein, streptavidin. Each protein molecule was labelled with 85 fluorescein molecules, and 50% of the proteins in the hydrogel were released after UV exposure for ∼100 s. The proteins released from the hydrogel were captured in biotinylated microtitre plates and detected by fluorescence, allowing measurement of at least 0.01 ppm (or ∼166 pM) of protein in sample solutions. The reported hydrogel is promising for detection of low abundance proteins while being less laborious than enzyme-linked immunosorbent assay and less affected by changes in environmental conditions than label-free biosensors.We report a novel hydrogel for pre-concentration, fluorescent labelling, and light-triggered release of proteins for detection of low abundance biomarkers. The hydrogel was a co-polymer of acrylamide/bisacrylamide and methacrylamide attached to fluorescein isothiocyanate  We report a novel hydrogel for pre-concentration, fluorescent labelling, and light-triggered release of proteins for detection of low abundance biomarkers. Measurement of low abundance proteins is key for enabling early detection of diseases.6 A widely used method for the measurement of low abundance proteins is enzyme-linked immunosorbent assay (ELISA).7 ELISA uses two antibodies where a capture antibody allows a target protein to be pulled out of samples and a detection antibody is labelled with an enzyme. A substrate is then added, which is acted upon by the enzyme to produce a fluorescent or coloured product. The fluorescence intensity or absorption is proportional to the concentration of the target protein. Furthermore, as each enzyme can act upon multiple substrate molecules, the signal is amplified, allowing measurement of low abundance proteins. ELISA, however, requires multiple adding and washing steps, and hence is laborious. The technique can be automated, but robotic handling systems are large and expensive. To address this challenge, ELISA has been implemented on microfluidic paper-based analytical devices by controlling the flow rate and hence arrival times of reagents to the detection zone.8 The flow control requires patterning of paper and multiple layers which must be precisely aligned. Alternatively, label-free biosensors have been used to measure low abundance proteins. Unlike ELISA, label-free biosensors9 require only capture antibodies and rely on changes in refractive index,10–12 impedance13,14 or mass15,16 to determine the concentration of target proteins. However, label-free biosensors are prone to errors because of changes in environmental conditions such as temperature.17 To measure low abundance proteins using absorption or fluorescence, which are less sensitive but require simpler instrumentation and are more user-friendly than ELISA, proteins have been pre-concentrated using beads18 and electrophoresis.19–21 After pre-concentration, proteins are recovered, labelled with dyes or fluorophores, captured, and then detected. This implies that a series of disparate steps are currently needed, much like ELISA.Much of the work on entrapment and release of molecules from hydrogels has been concerned with control of drug delivery.22 charged groups,23 cyclodextrins,24 nanoparticles25 and molecular imprinting26 to concentrate proteins in hydrogels. Release of the captured proteins has been by changing the swelling of the hydrogel by pH,3 temperature change1,2 or by degradation of the hydrogel using light27–29 or chemically.30,31 For example, Douglas et al.22 used N-isopropylacrylamide (NIPAm) – acrylic acid (AAc) copolymer microparticles modified by a dye (Acid black 48) covalently bound to the hydrogel. The dye acted as an affinity reagent to non-covalently bind proteins. Although this approach preconcentrated proteins from urine, it did not label them on release, the microparticles were extracted from the sample by centrifugation and the release was performed using an elution buffer which diluted the captured proteins. Detection of the captured and released proteins was by western blotting. The use of the microparticles for detection of proteins characteristic of Lyme disease did, however, improve the limit of detection by a factor of 10, from 0.07 ppm without microparticles to 0.007 ppm using microparticles.Previous work on the use of hydrogels for analysis has used non-covalent interactions with immobilised dyes,32,33 present in the hydrogels. As FITC is fluorescent, proteins were pre-concentrated and labelled in a single step. Furthermore, as shown in via a photolabile group, o-nitrobenzyl,34–40 which exhibits controllable photoreactions with tunable absorption for wavelengths >300 nm.38,40 Thus, the release of the fluorogenically labelled proteins was triggered through the irradiation of the hydrogels with UV light (365 nm). As shown in Herein, we report a novel hydrogel for pre-concentration, fluorescent labelling, and light-triggered release of proteins. The pre-concentration was achieved by covalent capture of proteins in hydrogels having much smaller volumes than sample solutions. As shown in To demonstrate the feasibility of our designed hydrogel for pre-concentration, labelling, and controlled release, streptavidin was used as an exemplar protein. The streptavidin used in this work was tagged with rhodamine so that fluorescence of rhodamine could be used to determine the protein concentration before and after incubation with our hydrogels. This in turn provided the pre-concentration factor. The designed hydrogels offered a pre-concentration factor of up to 236. Equally, the rhodamine label was used to study the light-triggered release kinetics of streptavidin from the hydrogels. We showed that 50% of streptavidin was released from our hydrogels in ∼100 s. Finally, we showed that released streptavidin was labelled with 85 fluorescein molecules per molecule of the protein. The designed hydrogel when combined with capture of released streptavidin using biotin and fluorescence detection, allowed detection of at least 0.01 ppm (or ∼166 pM) of the protein.2.1Mn 400 and 3400), 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butanoic acid (NVOC), (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolopyridinium 3-oxide hexafluorophosphate (HATU) (97%), N,N-diisopropylethylamine (DIPEA) (98%), triethylamine (TEA) (99%), methacryloyl chloride (97%), phosphate-buffered saline , phosgene solution (15 wt% in toluene), silica gel , sodium sulphate, methanol, ethyl acetate, hexane, DMSO-d6 , CDCl3 , and acetone-d6  were bought from Merck. Decon 90 was bought from Fisher Scientific. The remaining solvents were bought from Merck and prepared before use. Tetrahydrofuran (THF) and toluene were distilled under nitrogen from sodium using benzophenone as indicator. Dichloromethane (DCM), dimethylformamide (DMF) and acetonitrile were distilled under nitrogen from calcium hydride. Streptavidin tagged with rhodamine (S6366) was bought from Thermo Fisher.Allyltrichlorosilane (95%), acrylamide/bis-acrylamide , ammonium persulfate (≥98%), ethanol , fluorescein, fluorescein isothiocyanate isomer I (FITC) (≥90%), poly(ethylene glycol) (PEG) bis(amine)  were used for purification of PEG 3400 monomers. Pierce™ Biotin Coated Plates  were used for protein release studies. Microscope glass slides (1 mm thick low iron standard) were purchased from VWR .1H and 13C NMR spectra were obtained at 25 °C and recorded on a 300 MHz Varian VNMRS, 400 MHz Varian Unity Inova or 600 MHz Varian Unity Inova. Chemical shifts (δ) are reported in parts per million (ppm), and splitting patterns are designated as s (singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quartet), m (multiplet) and br s (broad singlet) and coupling constants (J) are expressed in Hertz (Hz). 1H and 13C NMR spectra are referenced to the residual solvent signal: DMSO-d6 (2.50 or 39.52 ppm) and CDCl3 (7.26 or 77.16 ppm).−2. The LED and DC-DC converter components driving the LED were mounted on a 60 × 60 mm2 aluminum–cored PCB, which in turn was mounted to a 60 × 60 mm2 heat sink and fan  using thermally conductive paste. The LED driver and fan were powered by a 12 V 1A DC mains adaptor.Irradiation at 365 nm was carried out using an in-house constructed source based on a UV LED  with a radiant flux of 1030 mW at 1A forward current expanded to 25 mm diameter, giving a flux density of ∼210 mW cmλexcitation = 472 nm, emission measured = 500–620 nm; rhodamine λexcitation = 540 nm, emission measured = 570–670 nm).UV–Vis spectroscopy measurements were carried out on a Jenway 6715 UV–Vis spectrometer at 25 °C. High-performance liquid chromatography (HPLC) was performed using Agilent 1260 Infinity coupled with a Vanquish™ multi wavelength absorbance detector and in a C18 column . A CLARIOstar Plus microplate reader was used to measure the fluorescence intensity of excited samples -NVOC-PEG(400 or 3400)-methacrylamide dissolved in 250 μL of deionized water, 40% w : v acrylamide/bis-acrylamide , 1.25 μL of TEMED, and 12.5 μL of 10% w : v APS were prepared in N2-degassed water. The concentration of the NVOC containing monomer was 5, 10, or 15% of the total molar concentration of the monomers in the precursor solution. The precursor solution was cast between glass squares and plastic covers separated with a spacer of 175 μm thickness  at room temperature in darkness. After 15 min, plastic covers were removed, and hydrogel films deposited on glass substrates were soaked in PBS overnight to remove any residual unreacted monomers.Glass microscope slides were cut into 25.4 ± 0.5 mm2.3−1 monomer stock solutions were prepared in acetonitrile and collected samples solutions were chromatographed using a solvent gradient of acetonitrile/water from 90/10 to 10/90. The chromatograms were recorded at a wavelength of 491 nm. The HPLC data was used to determine the kinetics of light-triggered release of fluorescein from monomers.The UV-Vis absorption spectra of monomers were measured at different concentrations to determine their molar extinction coefficient. Monomer stock solutions were irradiated with 365 nm light and samples were taken at fixed time intervals for subsequent analysis by UV-Vis spectroscopy and HPLC. For HPLC, 1 mg mlThe UV-Vis absorption spectra of a hydrogel film  in PBS were measured. Hydrogel films of acrylamide/bisacrylamide without and with FITC-NVOC-PEG3400-methacrylamide were submerged in a 10 mL stock solution of 0.01 or 0.1 ppm RS in PBS overnight. Fluorescence emission spectra of PBS before and after incubation with each type of hydrogel films were measured. The data was used to determine the factor by which proteins were pre-concentrated.Protein studies were carried out using functionalised hydrogels made by co-polymerizing FITC-NVOC-PEGThe hydrogel films were washed in fresh PBS at room temperature in dark conditions overnight to remove any unbound RS. Subsequently, the films were immersed in 3 mL PBS and exposed to UV-light irradiation in short time bursts of between 1 and 10 min. After each irradiation, the hydrogel was left in PBS in darkness for 30 min to allow the released RS to diffuse out of hydrogel films, and then the PBS was decanted from the hydrogel. The hydrogel films were then immersed in 3 mL of fresh PBS and the above process was repeated. The light-triggered release kinetics of RS was studied by monitoring the fluorescence emission corresponding to rhodamine with irradiation times. All measurements were performed in triplicate. To verify that the released RS is labelled with both rhodamine and fluorescein, 200 μl of the PBS solution collected after UV-light exposure was pipetted in a biotin coated well of a microtiter plate and incubated for 30 min. Subsequently, the well was washed with 200 μL of PBS and the fluorescence emission spectra were measured at excitation wavelengths of 470 and 540 nm corresponding to fluorescein and rhodamine, respectively.3.1400-methacrylamide and F-NVOC-PEG3400-methacrylamide monomers were water soluble and absorbed at wavelengths of ∼490 nm and ∼365 nm, which was attributed to fluorescein and NVOC groups.The chemical structures of the synthesized monomers are provided in 43–45 in this case, NVOC. Thus, the monomers solutions were irradiated to 365 nm UV-light. Typical UV-Vis spectra of the monomers for different irradiation times . Plots of ln[M]0/[M]tversus irradiation time  and precursor solutions (mprecursor). The mhydrogel and mprecursor were estimated based on the absorbance of the hydrogel and precursor solutions at ∼490 nm and using the molar extinction coefficient of fluorescein  and did not require purification by flash chromatography. Furthermore, the kinetics of the release of fluorescein in solutions of F-NVOC-PEG3400-methacrylamide was the fastest. Finally, the percentage of monomer incorporated in the hydrogel with respect to the precursor solution was higher for F-NVOC-PEG3400-methacrylamide than F-NVOC-allylamide . Considering these benefits offered by F-NVOC-PEG3400-methacrylamide, the protein studies were carried out using a derivative of this monomer, FITC-NVOC-PEG3400-methacrylamide, with an isothiocyanate group that could react with the primary amines in proteins.In summary, F-NVOC-PEG3.3Rhodamine–streptavidin (RS) was selected as an exemplar protein to study pre-concentration, labelling and release of proteins followed by their quanttation using fluorescence in biotin coated microtiter plates. The streptavidin used in this work was tagged with rhodamine so that fluorescence of rhodamine could be used to determine the protein concentration before and after incubation with hydrogels. This in turn provided the pre-concentration factor. Streptavidin was chosen because of its very strong and selective binding to biotin, which made it easy to capture in biotinylated microtiter plates after being released from hydrogels.3.3.13400-methacrylamide monomers, were capable of protein capture because the isothiocyanate groups reacted with the terminal amine and primary amines in proteins.47 This was confirmed by submerging the formed hydrogels in 10 mL of 0.10 and 0.01 ppm  RS solutions in PBS. As shown in The functionalized hydrogels, which were prepared by co-polymerizing acrylamide/bisacrylamide and FITC-NVOC-PEG40 where proteins were trapped inside hydrogels as a result of decreased pore size arising from electrostatic interactions, where a protein loading of 53.2 ± 7.4% was reported. Subsequently, the concentration of RS in functionalized hydrogels was estimated and the results are provided in Six functionalized hydrogels were prepared; three were incubated with 0.1 ppm RS solutions and the remaining three with 0.01 ppm RS solutions. As summarized in 3.3.2Having demonstrated the loading of a model protein into the prepared hydrogels, the photolytic release of protein from the hydrogels was investigated. The loaded hydrogels were exposed to UV in short bursts of between 1 and 10 min while submerged in 3 mL PBS, kept in PBS for 30 min to allow for the released RS to diffuse out of the hydrogel, and then the PBS was collected. This process was repeated until no additional RS could be detected in the supernatant PBS.r = 0.966), but a double exponential rise to a maximum gave a good fit with a correlation coefficient of 0.9995, indicating that two processes take place during the photolytic release of captured RS. The first fast process accounts for about 54% of the RS released with a time constant of 1.124 min−1, while the second slower process accounts for the remaining 46% of the released RS with a time constant of 0.075 min−1. From this we can show that 50% of the RS was released in ∼1.68 minutes, or ∼100 s. The origin of these two processes is currently unclear but may be a result of slow diffusion of released RS from the deeper layers of the hydrogel.The fluorescence emission spectra of the PBS collected after each irradiation are shown in 3400 spacer arm, making it easier for many FITC molecules to react with each RS molecule loaded in the hydrogels. The labelling of RS with large numbers of fluoresceins is beneficial for measurement of low abundance proteins.One of the PBS solutions containing labelled protein collected from a hydrogel incubated with 0.01 ppm RS and exposed to UV light was dispensed into a biotin coated well of a microtiter plate. The released streptavidin was allowed to bind to biotin for 30 min and then a buffer wash was performed. This process allowed removal of any fluorescein dye, not bound to protein but released when the hydrogel was irradiated with UV-light. The well of the microtiter plate was illuminated with 470 and then 540 nm light. The resulting fluorescence spectra see  showed evia a light cleavable bond achieved using o-nitrobenzyl (NVOC). The polymerizable group was either allylamide or methacrylamide. A poly(ethylene glycol) (PEG) spacer arm may or may not be present between the NVOC and the polymerizable group. We showed that the incorporation of methacrylamide monomer with 3400 g mol−1 molecular weight PEG spacer arm (F-NVOC-PEG3400-methacrylamide) in hydrogels was ∼65%, which was double that of the allylamide monomer without PEG. The other benefits offered by the F-NVOC-PEG3400-methacrylamide were water solubility, ease of preparation with high yield, and the fastest light-triggered release kinetics.This work reports a hydrogel for pre-concentration, labelling, and controlled release of proteins for their subsequent detection by fluorescence. The hydrogel was formed by co-polymerizing acrylamide/bisacrylamide with designed monomers. The designed monomers comprised of either fluorescein (F) or fluorescein isothiocyanate (FITC) attached to a polymerizable group By replacing fluorescein with FITC in the monomers and subsequently the hydrogels, a model protein was captured by reaction of amines on the protein with the isothiocyanate group. The designed hydrogels offered a pre-concentration factor of 192 and 236 for 0.1 and 0.01 ppm of an exemplar protein, streptavidin. Once pre-concentrated, the proteins were released by UV irradiation, leaving a free protein with a fluorescein label. We showed that 50% of streptavidin was released from our hydrogels in ∼100 s and was labelled with 85 fluorescein molecules per one molecule of the protein. The labelling of proteins with large numbers of fluorophores can allow measurement of proteins present at low levels. Finally, the designed hydrogel when combined with capture of released streptavidin using biotin and fluorescence detection, allowed detection of at least 0.01 ppm or ∼166 pM of the protein.The hydrogels developed in this work are promising for protein preconcentration, labelling and on-demand release. The developed hydrogel when used in combination with selective capture of labelled proteins and fluorescence detection offer potential for measurement of low abundance proteins in clinical samples, enabling early detection of diseases.Gupta did conceptualization, funding acquisition, project administration, resources, supervision, writing – review and editing. All other activities were done by both authors. All authors have given approval to the final version of the manuscript.There are no conflicts to declare.AN-148-D3AN00811H-s001


Cardiac troponins and NT-proBNP are biomarkers of cardiac injury that are used clinically in the diagnosis of myocardial infarction and heart failure. It is not known whether the amount, types and patterns of physical activity (PA) and sedentary behaviour are associated with levels of cardiac biomarkers.n = 2,370, 51.3% male, 28.3% T2D) we determined cardiac biomarkers hs-cTnI, hs-cTnT, and NT-proBNP. PA and sedentary time were measured by activPAL and divided into quartiles [quartile 1 (Q1) served as reference]. The weekly pattern of moderate-to-vigorous PA  and coefficient of variation (CV) was calculated. Linear regression analyses were conducted with adjustment for demographic, lifestyle, and cardiovascular risk factors.In the population-based Maastricht Study  and sedentary time on the one hand and hs-cTnI and hs-cTnT on the other. Those with the highest levels of vigorous intensity PA had significantly lower levels of NT-proBNP. With regard to PA patterns, weekend warriors and regularly actives had lower levels of NT-proBNP but not with hs-cTnI and hs-cTnT (reference:insufficiently actives). A higher weekly moderate-to-vigorous PA CV (indicating more irregular activity) was associated with lower levels of hs-cTnI and higher levels of NT-proBNP, but not with hs-cTnT.In general, there was no consistent association between PA and sedentary time and cardiac troponins. In contrast, vigorous and possibly moderate-to-vigorous intensity PA, especially if done regularly, were associated with lower levels of NT-proBNP. AddiPhysical activity is a well-known means to improve cardiovascular function and longevity . NeverthIndividuals with type 2 diabetes have higher levels of cardiac biomarkers compared with individuals with normal glucose metabolism which, in general, are also associated with adverse cardiovascular outcomes –19. HoweWe hypothesised that physical activity is inversely associated with cardiac biomarkers and sedentary time is positively associated with cardiac biomarkers and if the association is stronger in individuals with type 2 diabetes. In the view of the above, we investigated the relationship between the amount of physical activity in different intensities, and the pattern of physical activity over the week and sedentary time on the one hand and cardiac biomarkers on the other in a large population-based cohort with an oversampling of type 2 diabetes.2.2.1.We used data from the Maastricht Study, an observational prospective population-based cohort study. The rationale and methodology have been described previously .via mailings. Recruitment was stratified according to known type 2 diabetes status, with an oversampling of individuals with type 2 diabetes, for reasons of efficiency. This study included cross-sectional data from 3,451 participants, who completed the baseline survey between November 2010 and September 2013. The examinations of each participant were performed within a time window of three months.In brief, the study focuses on the etiology, pathophysiology, complications, and comorbidities of type 2 diabetes and is characterized by an extensive phenotyping approach. Eligible participants were individuals between 40 and 75 years of age and living in the southern part of the Netherlands. The population consisted mainly of Caucasian participants (>98%). Participants were recruited through mass media campaigns and from the municipal registries and the regional Diabetes Patient Registry The study was approved by the institutional medical ethical committee (NL31329.068.10) and the Minister of Health, Welfare, and Sports of the Netherlands (permit no. 131088-105234-PG). All participants gave written informed consent.2.2.2.2.1.Fasting blood samples were collected from all participants and processed according to the manufacturers' instructions. Serum samples were stored at −80°C for 1–4 years. High-sensitivity cardiac troponin T (hs-cTnT) was measured on a Roche Cobas 6000 analyzer (Roche) with the Elecsys Troponin T hs assay (Roche), which has a limit of blank (LoB) of 3 ng/L and a limit of detection (LoD) of 5 ng/L, and achieves a 10% coefficient of variation (10% CV) at 13 ng/L. High-sensitivity cardiac troponin I (hs-cTnI) was measured on an Architect i2000 SR analyzer (Abbott Diagnostics) with the Architect STAT High Sensitive Troponin-I assay (Abbott), which has an LoB range of 0.7–1.3 ng/L and an LoD range of 1.1–1.9 ng/L, and achieves a 10% CV at 4.7 ng/L. N-terminal pro-B-type natriuretic peptide (NT-proBNP) was assessed on a Roche Cobas 6000 analyzer (Roche) with the Elecsys proBNP II assay (Roche), which has a LoD of 5.0 ng/L (LoB not reported), and achieves a 20% CV at 50.0 ng/L. NT-proBNP and hs-cTNT concentrations below the LoD or LoB respectively, were set at LoD/2 and LoB/2, respectively .2.2.2.Physical activity and sedentary time were measured using the activPAL3™ physical activity monitor . The activPAL3 is a small (53 × 35 × 7 mm), lightweight (15 g) triaxial accelerometer that records movement in the vertical, anteroposterior and mediolateral axes, and also determines posture  based on acceleration information. The device was attached directly to the skin on the front of the right thigh with transparent 3M Tegaderm™ tape, after the device had been waterproofed using a nitrile sleeve. Participants were asked to wear the accelerometer for eight consecutive days, without removing it at any time. To avoid inaccurately identifying non-wear time, participants were asked not to replace the device once removed. Data were uploaded using the activPAL software and processed using customised software written in MATLAB R2018b  . Data frWe calculated the amount of time per week spent in light intensity physical activity (defined as standing and <100 steps/min), moderate-to-vigorous physical activity (defined as ≥100 steps/min), and vigorous intensity physical activity (defined as ≥130 steps/min) , 24. Totp < 0.001) and the mean difference in waking time between both methods was 0.02 h (1.2 min), with limits of agreement of −1.1–1.2 h.The total amount of sedentary time was based on the sedentary posture (sitting or lying), and calculated as the mean time spent in a sedentary position during waking time per day. The method used to determine waking time has been described elsewhere . In brie2.2.3.Covariates which were extracted from questionnaires included sex, age, level of education, smoking status, Dutch healthy diet index, mobility limitation, and history of cardiovascular disease. Level of education was categorized into low, medium, and high, and smoking status was categorized into never, former, and current smoker. Dietary habits were obtained from a validated food frequency questionnaire  and calcEstimated glomerular filtration rate (eGFR) was estimated with the CKD-EPI equation based on the combination of serum creatinine and serum cystatin C. To assess urinary albumin excretion, participants were requested to collect two 24-h urine collections. UAE was preferably based on the average of two (available in 91.9% of the participants) 24-h urine collections .2.3.p-values were calculated by anova (continues) or χ2 test .All data were analysed using IBM SPSS software version 26.0 for Windows . Characteristics of the total study population and according to moderate-to-vigorous physical activity patterns were summarized as mean (SD), as percentages or as median [interquartile range] (in case of a skewed distribution). The cardiac biomarkers were positively skewed and log transformed for all analyses described below. Associations between physical activity , sedentary time and physical activity pattern (independent variables) and cardiac biomarkers (dependent variables) were examined with the use of multivariable linear regression models. Physical activity and sedentary time of the total population were divided into quartiles due to non-linear associations. These non-linear associations were also found with restricted cubic splines , Q2: −0.11 ; 3.2.In the fully adjusted model, individuals in quartile 2 of total physical activity had statistically significantly lower levels of hs-cTnT compared to individuals in quartile 1 [Q2: −0.11 ; 3.3.In the fully adjusted model, individuals in quartile 4 of vigorous intensity physical activity had statistically significantly lower levels of NT-proBNP compared to individuals in quartile 1 [Q4: −0.12 ; 3.4.To investigate the mutual independence of the above associations, we adjusted light intensity physical activity and sedentary time for moderate-to-vigorous physical activity, and moderate-to-vigorous and vigorous physical activity for sedentary time. In general, the results were consistent with previous results , model 4After adjustment for demographic, lifestyle and cardiovascular risk factors, the physical activity patterns weekend warriors and regularly actives were inversely associated with NT-proBNP. There was no association between the physical activity pattern and hs-cTnI and hs-cTnT. For the coefficient of variation, there was an inverse association with hs-cTnI and a positive association with NT-proBNP, but not with hs-cTnT , model 3Interactions were tested in the fully adjusted model (model 3). There was no consistent statistically significant interaction with glucose metabolism status and sex (data not shown).To test the robustness of the above results, we did several sensitivity analyses. In general, results remained similar when we replaced BMI by waist circumference  and when4.This cross-sectional population-based study on the associations between accelerometer-measured physical activity and sedentary time on the one hand and cardiac biomarkers on the other had three main findings. First, there were no consistent associations between amounts or patterns of physical activity or sedentary time and levels of cardiac troponins. Second, the highest, as compared to the lowest quartile of vigorous and possibly moderate-to-vigorous physical activity was associated with lower levels of NT-proBNP, as was the pattern of moderate-to-vigorous physical activity: weekend warriors and regularly actives had significantly lower levels of NT-proBNP compared to the insufficiently actives. Additionally, there was an association between the CV and NT-proBNP, implying that less regularity of moderate-to-vigorous physical activity during the week was associated with higher levels of NT-proBNP. Third, associations in people with type 2 diabetes were similar to those in people without type 2 diabetes.It is well-known that acute bouts of physical activity can induce cardiac troponin release –34. HoweIn contrast, the highest quartile of vigorous (Q4 corresponds to >11.4 min/day) and possibly moderate-to-vigorous intensity physical activity was associated with lower levels of NT-proBNP. Earlier research reported an association between moderate physical activity and NT-proBNP , 37; ourPeople with type 2 diabetes, as compared to those without, usually have higher levels of biomarkers of cardiac injury –19, whicThis study had several strengths. We studied a large population-based sample with an oversampling of type 2 diabetes, so that we could study whether there was a difference between people with and without type 2 diabetes. Extensive measurements of physical activity were included  measured by a thigh-worn accelerometer. We used multiple cardiac biomarkers that were measured by appropriate techniques. We also adjusted extensively for accurately measured possible confounders, which makes unmeasured and residual confounding less likely. The study also had several limitations. For the calculation of physical activity pattern, week and weekend days were not weighted. Causal inference should be made with great caution due to the cross-sectional design. Additionally, the population consisted mainly of Caucasian participants, which limits generalisability of our findings. Due to statistical reasons (non-linearity), we categorized physical activity and sedentary time into quartiles, which entails loss of information compared to analyses that use all (continuous) data. Finally, unmeasured or residual confounding cannot be definitively excluded.In conclusion, our study shows that, independently of demographic, lifestyle and cardiovascular risk factors, in general there is no association between physical activity and sedentary time and cardiac troponins. In contrast, vigorous (>11 min/day) and possibly moderate-to-vigorous (>69 min/day) intensity physical activity, especially if done regularly, is associated with lower levels of NT-proBNP.


We report the case of nephrotic syndrome after COVID-19 vaccination. The patient was a man in his 30s with no comorbidities other than atopic dermatitis. Over the course of 2 weeks after the first COVID-19 vaccination, systemic oedema gradually appeared. He was referred to the nephrology department for investigation of the systemic oedema. On admission, he presented with pitting oedema in his lower extremities. Initial examinations revealed massive urinary protein and decreased serum albumin, at 13.9 g/g Cr and 1.5 g/dL, respectively. Renal biopsy was performed, and minimal change disease was diagnosed. Prednisolone 60 mg/day was promptly started on the 5th day of hospitalisation, and complete remission was achieved on the 12th day. Prednisolone was once tapered off in 1.5 years successfully though minimal change disease was relapsed in 1 month after the steroid withdrawal. SARS-CoV-2 infection and the resulting COVID-19 have caused extensive morbidity and mortality worldwide in the last 3 years.New-onset minimal change disease (MCD) requiring treatment after COVID-19 vaccination has been reported previously. MCD is the leakage of large amounts of protein into the urine due to increased permeability of the glomerular filtration barrier. The involvement of vaccination in the onset and recurrence of MCD has been known, and most cases develop within 2 weeks after vaccination. Thirty cases of new-onset MCD after COVID-19 vaccination have been reported worldwide, including 16 cases related to the Pfizer-BioNTech vaccine, 5 cases related to the Moderna vaccine, 5 cases related to the AstraZeneca vaccine, 2 cases related to the Sinovac vaccine and 2 cases related to the Janssen vaccine.In this case report, we present a case of new-onset MCD following the first injection of the Moderna mRNA-1273 SARS-CoV-2 vaccine. We successfully maintained remission with steroid monotherapy and completely terminated steroids after 1.5 years. We report our findings with pathology and literature review.2. The day after the patient received the first dose of the Moderna mRNA-1273 SARS-CoV-2 vaccine, he experienced low-grade fever and swelling at the site of the injection. The fever and local swelling improved after 1 day, but leg oedema appeared 2 days after vaccination. Over the course of 2 weeks, generalised oedema, palpitations and shortness of breath gradually worsened. He visited a local doctor and was prescribed a diuretic, but the symptoms did not improve. Therefore, 15 days after the vaccination, the patient was referred to our department and was admitted that day.The patient was a man in his 30s with a history of atopic dermatitis and obesity with a body mass index of 31 kg/mOn admission, his blood pressure was 142/93 mm Hg and heart rate was 81 beats per minute. His body weight had increased from 90 kg to 101 kg with systemic oedema. Blood tests revealed serum creatinine 0.85 mg/dL, blood urea nitrogen 17.2 mg/dL, serum albumin 1.5 g/dL, Low Density Lipoprotein (LDL) cholesterol 363 mg/dL. In addition, a high degree of proteinuria with urinary protein-creatinine ratio (UPCR) of 13.9 g/g Cr was observed, meeting the diagnostic criteria for nephrotic syndrome. A 24-hour urinary collection revealed proteinuria of 18 346 mg/day, and the selectivity index of urinary protein was 0.094. Antibody screening tests such as antinuclear antibody, antineutrophil cytoplasmic antibody and antiglomerular basement membrane were all negative. Total haemolytic complement (CH 50) and the serum complement levels of C3 and C4 were within normal range. Serum immunoglobulin (Ig) E level was markedly elevated at 2620 IU/mL. A PCR test for SARS-CoV-2 was negative. However, the SARS-CoV-2 anti-S antibody titre was 452 U/mL using the Elecsys Anti-SARS-CoV-2 S RUO (Roche Diagnostics K.K.) and 2650.3 AU/mL using the ARCHITECT SARS-CoV-2 IgG II Quant (Abbott Japan), which was markedly elevated. CT examination showed normal kidney size and ascites effusion.Renal biopsy was performed the day after admission. The histological findings are shown in The clinical course of this case is shown in On the 12th day, the UPCR decreased to 0.1 g/g Cr and complete remission was achieved. After the remission, time was required to manage the side effects of high-dose steroids , and the patient was discharged on day 34 of hospitalisation when the dose was reduced to moderate-dose steroids. Two months after discharge, serum creatinine was 0.62 mg/dL, serum albumin 4.6 g/dL and UPCR was 0.14 g/g Cr. The patient remained in complete remission for more than 1 year and PSL was completely tapered off approximately 1.5 years after the onset. However, 1 month after completion of PSL treatment, a relapse of nephrotic syndrome was observed, so PSL 20 mg was restarted, and remission induction was achieved quickly. The second vaccination was not administered based on consultation with the patient. We suggested that if the patient wanted to be vaccinated in the future, another type of COVID-19 vaccine, such as inactivated vaccine, could be administered, but the patient did not wish to be vaccinated.This case highlights the risk of new-onset MCD after mRNA vaccine administration. Several case reports in regard to the association of MCD with the Moderna mRNA-1273 SARS-CoV-2 vaccine have been published previously. Full case reports of new-onset MCD cases after Moderna mRNA-1273 SARS-CoV-2 vaccination are few and no reports mention long-term outcomes. In MCD, a state of hyperpermeability of glomerular capillaries occurs, resulting in highly selective proteinuria. Although the exact pathogenesis has not yet been elucidated, injury of podocytes by circulating factors released by T cells is thought to be the main mechanism.The inevitable question is whether the timing of COVID-19 vaccination and the onset of nephrotic syndrome were too close together. The timing of MCD onset or relapse was reported at a median of 7 days following the first dose of the COVID-19 vaccine.et al reported a case of new-onset MCD following the Pfizer-BioNTech SARS-CoV-2 vaccine in a 4os woman with asthma.In combination with COVID-19 vaccination, atopic disease may have predisposed this patient to developing MCD. Pre-existing atopic dermatitis has been associated with the development of MCD through IgE-mediated hypersensitivity, which could imply that those with atopic dermatitis become more susceptible to potential allergic stimulation such as mRNA vaccination, leading to MCD, than those without atopic dermatitis. Additionally, MCD is frequently observed in patients with bronchial asthma and atopic dermatitis, and the relationship between IgE-mediated type I allergy and MCD has been highlighted.Steroid sensitivity of new-onset MCD after mRNA vaccination is often good, and there are no case reports of spontaneous remission. Most previously reported cases without underlying disease describe remission induced with steroids alone.Although the causal relationship is uncertain, minimal change disease following mRNA vaccination has been reported.As a possible pathogenic mechanism, the mRNA vaccination may have affected the immune regulatory system, especially the T-cell profile, causing podocyte damage.In addition, the patient had atopic dermatitis as a background disease, and it is thought that the predisposing factor was an atopic disease characterised by a Th2-dominant immune state.